Team:Brown-Stanford/Lab/Notebook/Week8

August 1, 2011

PowerCell

• Picked colony of pDS4101 x pRL25 to liquid culture.
• Gibson of Ana med and Nos med (the cscB ttsp constructs! Maybe saw faint bands after loading 5ul of Gibson product
• Transformed 7.5ul of Gibson product into TOP10; plated on LB chlor plates (100ul on one plate, 200ul on another) at 7pm

• used assembly master mix and protocol described in “Gibson protocol-openwetware” file
• 15ul of master mix with above part amounts incubated at 50?C for 1 hour.
• Ran gel of backbone amplification, saw nothing
• Plasmid was at 5x concentration, 125ng/ul!!! add more water and try again.
• Innoculate liquid Nostoc cultures (2x 20ml cultures)
• Made LB+agar+chloramphenicol plates
• Redo PCR of plasmid backbonds pSB1A2, pSB1C3:
  • gradient from 40-50C
• imaged (CAUTION: the key for lane/samples may be reversed; ie pSB1A2 and pSB1C3 may be flipped; the description in the lab notebook is 75% certain)

gel extracted what is likely pSB1C3 (heavy band between 2000 and 2500bp ladder); results: 25ng/ul

August 1, 2011

REGOBricks

• First Transformation of S. pasteurii:
• Materials:
  • Cells (frozen cryostock of 1 mL: suspended in 18% glycerol and 1X Spizzen’s)
  • SMMP (+BSA)
  • SMMP
  • Lysozyme
  • screw-top test
  • centrifuge
  • 40% PEG
  • 2xSSM
  • 5 ug of transforming DNA, for each 1 mL aliquot of cells
  • vy-R5 regeneration plate
  • vy-R5 regeneration plate + Kan

• Procedure:

1. Cell Preparation:
   1. Day 1: Streak strain to be protoplasted and incubate at 25 C.
   2. Day 2:
      1. Different Day prep:
         1. inoculate 200 mL of MM2 (subtilis), Bang (pasteurii) in a 2 L flask. Incubate at 37, until OD reading of 0.2. Harvest at 4 degrees, 8000g (rcf) for 10 minutes. Wash cells with sterile distilled water. Resuspend cells in 6 mL of 1x Spizizen with 18% glycerol and freeze in 1 mL aliquots. Store at -80 C at least overnight.
      2. Same Day prep:
         1. inoculate 30 mL of MM2 (subtilis) or Bang (S. past) with cells from an overnight culture in a 300 mL. Incubate at 37 until an OD of 0.2. Harvest cells and resuspend in 4.5 mL of SSMP + BSA.
   3. Day 3:
      1. LYSOZYME
      2. Thaw a 1 mL aliquot, added to 3.5 mL of SSMP (+BSA) in a 125 mL flask
         1. Add 0.5 mL of 5 mg/ml lysozyme dissolved in SMMP + BSA (final concentration 0.5 mg/ml)
         2. (Wild type strains may require 10 ug/ml of lysozme)
         3. (if cells are in lysozyme >1 h, regeneration of cells is difficult)
         4. Incubate cells at 37 C and 75 rpm: remove a sample of 10 ul every 15 minutes, examine microscopically for extent of protoplastation
      3. TRANSFORMATION
         1. prepare protoplast suspension:
         2. transfer suspension to sterile 13 x 100 mm screw top test tube
         3. harvest cells by centrifuge (2580 g for 15 minutes) [clean out lysozymes]
         4. Decant, wash cells GENTLY with 5 mL of SSMP [wash out cells]
         5. Centrifuge at 2580 for 10 minutes
         6. Resuspend protoplast GENTLY with 1 mL SSMP
      4. DNA
         1. Add 5 ug of transforming DNA + equal volume 2xSSM to screwtop tube
         2. Add 0.5 mL of protoplast suspension
         3. IMMEDIATELY add 1.5 mL of 40% PEG (have another 1000 ul pipette ready in the hood, set to 750 ul, pipette twice)
         4. Invert solution for a minute
         5. add 5 ml of SSMP + BSA
         6. Mix gently and centrifuge*** for 5 minutes. Speed:
            1. • • • 40% PEG very viscous, makes pelleting by centrifuge difficult
               1. Tried 5 mins, 2580 - FAIL
               2. Tried 5 mins, 3000 - FAIL
               3. Tried 5 mins, 4000 - FAIL
            2. Tried 15 mins, 2580 - FAIL
               1. Tried 5 mins, 4500 - FAIL
               2. Tried 10 minutes, 4800- FAIL
         7. Resuspend pellet in 1 mL of SMMP + BSA
         8. incubate at 30 C for 90 minutes to allow for expression
         9. Recentrifuge and suspend in 1 mL SMMP + BSA

• Plating/Dilution
  • Set up 5 test tubes-

• Plate onto Regeneration plates
  • Plate onto transformation plates

• Possible selection of variation

1. Concentration of lysozyme (but it can be standardized by equal concentration of protoplasts as seen under the microscrope: According to Ryan, protoplast cells transform from rod shaped to spherical shape as they undergo protoplastation)
2. centrifuge speed- should redo at 2580 rcf, maybe sample protoplast under microscope to see extent of cell survival

• ELECTROPORATION
  • Dilute overnight culture 10 fold in neutral complex media (maybe just use BANG?)
  • Add glycine to a final concentration of 1% and grow for 1h a 37?C (Glycine can be varied)
  • Cool cells on ice for 10min
  • Centrifuge @ 4?C 5000xg for 5 min to collect cells
  • Wash 4x with electroporation media (wash-spin-wash-spin…)
  • Resuspend in electroporation medium
  • Can stop at this step and freeze @ -80?C
  • For electroporation, mix 60ul of cells with 2.5ul DNA and transfer to ice cold electro-cuvette
  • Incubate on ice water for 10 min
  • Expose cuvette to single pulse using Gene-PulserII set at 25uF and 200 ohms
  • Immediately after discharge, add 1ml of recovery medium to cells and shake at 37?C for 3h
  • Plate on BANG and BANG Kan plates

• Making Bricks:

• • Process:
    • Grow up S. pasteurii
    • Spin them down,
    • Resuspend them in three
    • Insert into column
    • Insert CaCl2
    • Wait- 7 days

• Variations:
  • Vary cells (S. past 11859 vs Dosier)
  • Vary concentrations of CaCl2, urea (1:1 M, 2:1 M; 2:2 M; 1:3)
  • Vary resuspension
  • Vary concentration of cells
  • How much cell we are putting in (volume)

August 2, 2011

PowerCell

• Check on plated Gibson products: no transformants :(
• Prepped all of our Gibson bits for sequencing
• cryostocked pDS4101 x pRL25
• prepare transformation of just pRL25, pDS4101, pRL443, Anabaena with serial dilutions.
• made plates (BG11+kan, BG11+LB, BG11) and E. coli liquid cultures (afternoon)
• liquid cultures of plasmid strains
• Gibson attempt #2, Evan.

• Incubation begun @ 11:20, take out @ 12:20.
• ran 5ul of Gibson products on gel; saw a collage of various band lengths (Lane 1: Ana Med w/ registry backbone | Lane 2: Ana M w/ PCR backbone | Lane 3: Nos M w/ reg backbone | Lane 4: Nos M w/ PCR backbone)
• Transformed 7.5ul of Gibson product into TOP10s, plated on chlor plates
• We are running out of Phusion-made cscB|TTSP! Maybe 8 uL left.

August 3, 2011

PowerCell

• Transformation
  • transformation of just pRL25, pDS4101, pRL443, Anabaena with serial dilutions.
  • balls! Kanamycin won't select for cyanobacteria, so neomycin is necessary. ordered overnighted, proceeding in the meantime. Neomycin plates are only necessary for selection of exconjugates 1-2 days into the process, so hopefully it’ll be here in time.
• Due to time constraints and demand for the hood, the plate mating protocol was followed, rather than spot mating. The nitrocellulose was wet.
• autoclaved nitrocellulose slips for transformation filters
• Found 1 colony from our Gibson Attempt (#2) Aug3 transformants, on the Anabaena-PCR-backbone plate! Innoculated liquid cultures in preparation for mini prep.
• left at room temperature until morning of 8/4, oops

Trying the transformation again with greater cells to DNA ratio. All 4 backbone/species combinations.

• PCR: cscB|TTsp, cscB|GFP linker, GFP ttsp, NosS
• left to run overnight
• Began to prepare materials for cyanos on balloon flight

FRETSensor

• New UV directed evolution testing:
  • Grow puc18 cells in 25 mL LB
  • Zap at 1 min, 5 min, 10 min in:
    • 1 mL LB exposed (a)
    • 1 mL PBS exposed (b)
    • plated on LB amp plates
  • Plate (a) and (b)
  • Zap amp plates 10 min, inoculate with top10, to see if it degrades
• Made competent E. coli W with CT2, transformed with RFP

August 4, 2011

PowerCell

• Transformation of just pRL25xpDS4101, pRL25, pDS4101, pRL443, Anabaena and Nostoc with Elhai’s plate transformation protocol. Incubating in 30?.
• Found weird mystery red colony on one of the Aug2 gibson attempt plates - took microscope pictures, they look like E. coli. Could they be cross-contamination from Lei and Jovian’s RFP strains? Those shouldn’t grow on our chlor plates...
• Going to pick colonies for liquid culture, mini-prep anyway, possible sequencing anyway.
• Also pick 4 more colonies from non-suspicious colony and grow up for liquid culture/miniprep, because we forgot to put the liquid cultures from yesterday in the incubator, they may not grow....
• cultures from 8/3 looking more promising
• Ran gels on PCR from 8/3 PM
• cscB|TTsp and cscB|GFP fail
• lanes cut and saved from GFP|TTsp and Nosstr
• Continue to prepare cyanos for balloon flight.
• redesign primers for bio brick assembly approach as alternative to gibson (Primers ordered from Elim! Melting temps between 55-57C)

FRETSensor

• Discovered that ampicillin does not degrade after 10 min of UV!
• LB media shields cells
• Plating directly onto LB amp plates seems to work best/easiest. No E. coli survived even 1 min.
• Plating from PBS, no e coli survived 1 min of UV
• Made liquid cultures of E. coli W w/ RFP for plasmid prep and replating
• GeneArt order #2 came in (where #1??) with the C. cellulolyticum Doc-YFP and C. cellulolyticum YFP-Doc. Unfortunately, they do not work with Chad’s rosettazyme, but we will try transforming and plasmid prepping today.
• RFP successfully transformed into E. coli W, plated on to BG11 sucrose plates with ampicillin resistance (5 mM, 10mM, 30mM, 60mM, 80mM, in increments of 50uL, 100uL, and 200uL)
• Spread some of the gel stab of the cohesin-cohesin construct on an LB amp plate

August 5, 2011

PowerCell

• Made BG11 + 1% agar + 50ug/ml neomycin plates
• Transferred conjugation filter from BG11+5%LB+1% agar plates to above neo plates.
• began construct design for synthesis. uploaded to construct design folder.
• Miniprep Gibson colonies
• worked on balloon experiment prep
• Chucked the first and second cyanobacteria transformation attempts.
• no cyanobacterial growth
• appearance of conjugative parents, numerous e. coli colonies
• Prepped Cyanos for balloon flight.
• Took hemocytometer/OD readings of Nos, Ana so we know how many cells we flew
• Anabeana: 1.4e6 cells/mL = .265 OD -> added 10 ul cells to nitrocellulose
• Nostoc: 1.1e7 cells / mL (can’t OD, too clumpy) -> added 10 ul cells to nitrocellulose

August 5, 2011

REGOBricks

• To Do List for friday 8/5/11

1. Look at ecoli transformation in 37C (no transformation - repeat)
   1. If there are colonies, inoculate LB amp liquid culture to harvest puBC3
   2. Look at s. pasteurii in 30C to see if recovery KAN plates actually have KAN (Did not grow, so plates are still okay? Will make new plates)
   3. growth = no KAN (either way probably make some new plates)
2. Make new recovery plates, w and w/o KAN
3. Find two tubes in 37 C, in a white tub on the lower level with some culture tubes there are two blue 1.5mL tubes labeled 1 and 2 (I know helpful right?), these are recovering from the

protoplast/electroporation protocol (its like these cells got their skin ripped off and then were electrocuted ha) They need to be plated on recovery plates both antibiotic and non

1. Make new recovery plates, w and w/o KAN
2. re-do protoplast transformation with good plates?
3. Be awesome.

FRETSensor

• GeneArt order #1 came in!!
• E. coli W has not grown on sucrose plates
• Gel stab overgrown